Electroporation with nanosecond pulses and bleomycin or cisplatin results in efficient cell kill and low metal release from electrodes.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.

https://www.pnas.org/content/pnas/118/45/e2110817118.full.pdf

An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.

Objective. To evaluate the effects of nanosecond pulsed electric fields (nsPEFs) with different pulse durations in cell vitality, apoptosis, and proliferation of TPC-1 cells, optimize pulse parameters and expand the application range of nsPEFs. Methods. The pulse duration of 0, 300 ns, 500 ns, and 900 ns is generated with nsPEF generator. CCK-8 was used to investigate the effect of nsPEFs on the viability of TPC-1 cells. Flow cytometry was used to evaluate the apoptosis of TPC-1 after pulse treatment. The effect of nsPEFs on the proliferation ability of TPC-1 cells was detected by 5-ethy-nyl-2 - deoxyuridine. The morphological changes of TPC-1 cells after pulse treatment were observed by transmission electron microscopy. Results. NsPEFs with 900 ns pulse duration can significantly affect the viability of TPC-1 cells and inhibit the proliferation ability of TPC-1 cells. In addition, nsPEFs can also induce apoptosis of TPC-1 cells. Conclusion. NsPEFs with longer pulse duration can significantly affect the biological behavior of TPC-1 cells, such as cell viability and proliferation ability, and can also induce cell apoptosis, thereby inhibiting cell growth.

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Effects of Nanosecond Pulsed Electric Fields in Cell Vitality, Apoptosis, and Proliferation of TPC-1 Cells.

this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses.As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 μL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 μs x 8 protocol was used.It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses.the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 μs pulses. 

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy

The increased research activity aiming at improved delivery of pharmaceutical molecules indicates the expansion of the field. An efficient therapeutic delivery approach is based on the optimal choice of drug-carrying vehicle, successful targeting, and payload release enabling the site-specific accumulation of the therapeutic molecules. However, designing the formulation endowed with the targeting properties in vitro does not guarantee its selective delivery in vivo. The various biological barriers that the carrier encounters upon intravascular administration should be adequately addressed in its overall design to reduce the off-target effects and unwanted toxicity in vivo and thereby enhance the therapeutic efficacy of the payload. Here, we discuss the main parameters of remote-controlled drug delivery systems: (i) key principles of the carrier selection; (ii) the most significant physiological barriers and limitations associated with the drug delivery; (iii) major concepts for its targeting and cargo release stimulation by external stimuli in vivo. The clinical translation for drug delivery systems is also described along with the main challenges, key parameters, and examples of successfully translated drug delivery platforms. The essential steps on the way from drug delivery system design to clinical trials are summarized, arranged, and discussed.

Key Points in Remote-Controlled Drug Delivery: From the Carrier Design to Clinical Trials.

Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment-electrochemotherapy. However, electrochemotherapy is associated with microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy.

https://www.mdpi.com/1420-3049/25/20/4601/pdf

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study.

The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1–5 μs × 250 and 100 μs × 1–8 pulsing protocols in 1–2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

Electroporation is a highly effective method in various fields of application, including cancer treatment, where when combined with drugs (called electrochemotherapy), it improves treatment via permeabilization of cells. In this work, we analyze current and voltage measurements deviation as an indicator of tumor permeability and conductivity changes in the nano (3.5 kV/cm × 800 ns × 250 × 4) and microsecond (1.4 kV/cm × 100 µs × 8) pulse ranges. The distribution of the electric field inside the tumor was approximated using a finite element method simulation in a COMSOL Multiphysics environment. Our results indicate that both nano and microsecond range pulses are suitable for effective electrochemotherapy, while measurement of the current and voltage deviation can serve as an indicator of successful cell membrane permeabilization.

Veronika Malysko

Papers

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

Measurement and Evaluation of Electric Pulse Parameters to Improve Efficacy of Electrochemotherapy